Certain microorganisms are natural pathogens/parasites for Japanese beetles, European chafers and related insects belonging to Family Scarabaeidae. There are over 19,000 species of beetles belonging to Family Scarabaeidae. Wherever the beetles occur in large populations, milky disease bacilli have been found naturally infecting portions of the populations. There are now about 12 distinct morphological types of bacilli that cause milky disease in one beetle species or another. They are all rod shaped in the reproductive stage and form spores in the resistant dormant stage. Some such as B. popilliae contain a parasporal body. Some do not contain parasporal body and, in some cases, the spore is smaller than a parasporal body in B. popilliae. In all cases, the spores are ingested by healthy larvae, germinate either in the gut or gut tissues possibly aided by lymphocytes, multiply in the tissues or in the hemolymph and eventually sporulate in the tissues or hemolymph. Only after cell/spore populations have reached to the billions/ml of hemolymph does the host die and release its spore load into the soil where the spores remain dormant (but alive) until consumed by a susceptible host to repeat the cycle. Scientists have been studying Bacillus popilliae and other milky disease bacilli for the last 40 to 45 years. It was recognized very early that these bacilli offered a potential method for biological control of the beetles. Dr. S. R. Dutky of the U.S.D.A. obtained a patent (U.S. Pat. No. 2,293,890) on a process of producing the spores of Bacillus popilliae by injecting the bacilli into Japanese beetle larvae, allowing the bacilli to complete their life cycle including formation of spores and then grinding the diseased larvae with a diluent such as talc. The resultant spore dust was inoculated into the soil and served as a means of infecting and controlling succeeding populations of Japanese beetles and European chafers that might have invaded the area. Further patents dealing with the production of B. popilliae as microbial insecticides includes U.S. Pat. Nos. 3,308,038; 3,503,851; 3,616,250 and 3,950,225. While spores produced by the in vivo process have been on the market for a number of years, the method is handicapped by the necessity of collecting and inoculating thousands of larvae each year.
For the past 25 to 30 years, scientists have been endeavoring to elucidate the factors controlling sporulation in vivo and particularly in vitro with the objective of developing methods of producing the spores en masse in vitro. Steinkraus and Tashiro (Science. 1955. Vol. 121:873-874) produced spores of B. popilliae in vitro by growing the vegetative cells on the surface of a suitable growth medium and then transferring the cells as a paste to the surface of a "starvation" medium on which further vegetative growth was impossible. While this in vitro method of producing spores yielded sufficient spores to establish that the spores were virulent when fed to or injected into healthy European chafer larvae, it was strain related, difficult to reproduce repeatedly on a large scale and therefore was impractical commercially.
Subsequently, the U.S. Department of Agriculture undertook an expanded research program on the in vitro production of spores of B. popilliae. The USDA developed strains of B. popilliae that formed spores in vitro under certain conditions. In all cases, particular strains were required. Some of the methods required the use of specific batches of ingredients. However, in contrast to the method of Steinkraus and Tashiro, above, either the USDA method failed to produce sufficient spores to test or when tested, the spores were no longer virulent per os. And, in no case, up to the present, (including the Steinkraus and Tashiro method) was the factor or factors controlling sporulation apparent.
The Steinkraus and Tashiro method depended upon cultivation of the vegetative cells and the sporulation phase both on the surface of solid media.
The USDA methods also used the surfaces of solid media in some cases but tried to cultivate the vegetative cells in submerged culture as this procedure is more adaptable to large scale commercial production of vegetative cells and spores. Unfortunately, in submerged culture, the cells of B. popilliae tend to grow to a peak population in less than 20 hours and then they die very rapidly. Dead vegetative cells, of course, will not sporulate.
R. Skole and A. B. Rizzuto obtained U.S. Pat. No. 3,950,225 which described a process whereby vegetative cells of B. popilliae grown on a suitable medium sporulated when suspended in bone char waste water. The present inventor has confirmed that vegetative cells derived from germinated spores removed from milky diseased larvae and grown on a suitable medium do sporulate when circulated through bone char columns under certain conditions. The factor or factors controlling sporulation in the bone char have not been elucidated. The mechanism could be removal of substances inhibiting sporulation or concentration of cells or substances required for sporulation or both. It was found necessary to grow the vegetative cells of B. popilliae on the surface of agar plates in pure culture, suspend them in a mineral salts medium that resembles char waste water and circulate them through the bone char column in order to obtain sporulation. Attempts to use submerged cultivation of the cells highlighted of the difficulty of maintaining cell viability from time of growth to time of circulation through the column. The Skole and Rizzuto patented method does not reveal the essential factor or factors controlling sporulation of B. popilliae.
Sporulation of milky disease bacilli in vitro has been studied intensively for 30 years, yet the factors controlling sporulation in vitro or in vivo remain unknown. Bennett and Shotwell (1970, J. Invert. Path. 15:157-164) reported that the content of C.sub.16 -C.sub.22 unsaturated fatty acids in hemolymph decreased markedly during the course of milky disease. They further suggested (J. Insect Physiology (1972) 18:53-62) that lipids might be altering membrane permeability (of the bacilli) and influencing enzyme activity related to sporulation. Bulla, Bennett and Shotwell (1970, J. Bact. 104:1246-1253) recognized that the lipid components might be playing an important role in growth and sporulation of B. popilliae but apparently none of the studies were tied directly into sporulation either in vivo or in vitro.
There is virtually nothing in the sporulation literature that would suggest that fatty acids might play an important role. In fact, there is evidence in the published literature that would suggest that fatty acids are inhibitory to sporulation, Humfeld (1974, J. Bact. 54:513-517) reported an "antibiotic-like" activity for C.sub.18 fatty acids extracted from wheat bran. Gram-positive cocci were inhibited but Gram-negative Escherichia coli was not. Foster and Wynne (1948, J. Bact. 55:495-50) reported that the C.sub.18 unsaturated fatty acids, particularly oleic inhibited growth of a wide-range of bacteria including Clostridium botulinum, an anaerobic sporeformer. They reported that small amounts of oleic, linoleic and linolenic acids inhibited germination of spores of C. botulinum and that addition of 0.1% soluble starch eliminated the inhibition. Wynne and Foster also reported that the addition of 0.1% soluble starch to pork infusion thioglycollate broth increased germination of C. botulinum thirty times.
The studies of Humfeld and Wynne and Foster are of interest in retrospect because it had been reported earlier (Lehrman, 1929) that rice starch contained 14.75 grams of mixed unsaturated fatty acids/5 lbs of rice starch; and Taylor and Lehrman reported (1926, J. Am. Chem. Soc. 48:1739-1743) that corn starch contains 0.5 to 0.6% unsaturated fatty acids.